Grain production

ABSTRACT

This invention describes a new method to increase grain yields in any crop plant by modifying pollination to effect an increase in grain yield, a change in grain content or characteristics, a decrease in contamination, or a combination of these attributes. The process involves the intentional delivery of pollen of the male plant at will, as available either in a preserved pollen bank, or real-time collection from male plants as they become available, in a growth chamber for example. Desired pollen is delivered to fertile females. The delivered pollen M  is in such amounts and fortuitously timed that it preferentially pollinates the females and optionally, avoids or enhances self-pollination or pollination from neighboring plants. The intentional delivery of genetically different pollen will result in increased heterosis and accompanying grain yield increases resulting from increased grain size and the potential to influence grain content and constituents. The invention also permits real-time agronomic decision making in order to maximize grain yield by overcoming biotic and abiotic challenges in the growing season which may or may not have been anticipated. The intentional delivery of self- or sib-pollen results in a decrease in contamination from undesirable outcrossing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/184,596 filed Jun. 25, 2015 and entitled SEED PRODUCTION, andfrom U.S. Provisional Application Ser. No. 62/269,496 filed Dec. 18,2015 and entitled SEED PRODUCTION, and from U.S. Provisional ApplicationSer. No. 62/269,531 filed Dec. 18, 2015 and entitled GRAIN PRODUCTION,and from U.S. Provisional Application Ser. No. 62/269,514 filed Dec. 18,2015 and entitled GRAIN PRODUCTION. The contents of U.S. ProvisionalApplication Ser. Nos. 62/184,596; 62/269,496; 62/269,531; and 62/269,514are hereby incorporated in their entireties by reference.

FIELD OF THE INVENTION

This invention relates generally to novel methods of grain production,specifically intended to increase grain yields. More specifically, thisinvention relates to on-demand pollination technology applicable tograin-bearing plants which is not dependent on active pollen shed,neighboring plants, or seed mixes.

BACKGROUND

The current invention has application to the field of grain productionand plant pollination practices, including, but not limited to,economically significant grain crops such as maize (also called corn),soybeans, wheat, rice, sunflower, canola, sorghum, barley, and pearlmillet. The term “grain production” as used in this disclosure isintended to mean commercial-scale grain production, using a minimumfield size of 0.8 hectares (2 acres) of land for the production of saidgrain. A conventionally grown grain field is typically predominantly F1plants that share genes in common from 2 inbred parents (althoughconventional grain will also have a small percentage of contaminant seed(not F1) in a standard bag of seed.). In other situations, the“top-cross” method is used for grain production. In contrast to theconventional field of F1 plants, top-cross grain uses a field that is ablend of a male sterile hybrid (˜93%) and a male fertile high oilpollinator germplasm (7%). Commercial grain production often relies uponfield production of hybrid plants. Hybrid plants are the result offertilization occurring from a male pollen source of one geneticbackground being crossed to the female reproductive organs of a plantwith a different genetic background. Hybridity among crop plantsgenerally gives a yield advantage in commercial production. Currently,maize, rice, sorghum, sunflower, and canola are the primary crops whichtake advantage of hybrid seed. Other grains are less commonly grown ashybrids, as is the case with soybean, wheat, barley, pearl millet andothers.

For crops in which grain production is commonplace, current methods ofproducing grain vary slightly by species, but typically involve plantingfields of the same seed variety to produce plants whose mature seedswill result in the desired grain. The plants in such fields aretypically self-pollinated or pollinated by neighboring plants in thefield. There may be some cross pollination from nearby fields of similarspecies.

For the purposes of this disclosure and its applicability to grainproduction, the term “self-pollen,” which is a single plant's ownpollen, includes “sib-pollen,” which is pollen from sibling plants whoshare the same genetics. Likewise, the term “self-pollination” includes“sib-pollination.” which is pollination occurring with pollen from asibling plant, and which has the same effect in the resulting grain asself-pollination. In a hybrid grain production field, allself-pollination and sib-pollination is effectively self-pollination interms of the effect on the grain, which is a yield decrease (incomparison to outcrossing) and purity increase in the case of food corn.“Cross pollination,” for the purposes of this disclosure, refers togenetic exchange between inbreds or hybrids in adjacent and surroundingfields, not from plants within the same field. Thus, cross pollinationis the introduction of pollen that is derived or sourced from separatelocations that is genetically distinct from the pollen which will beshed from the plants within the grain production field.

The advantage of using hybrid seed for grain production is that hybridsare known to exhibit heterosis, which is expressed as hybrid vigour,meaning a stronger and more resilient plant. Heterosis also results inhigher grain yields. This effect is sometimes referred to as xenia.(Stamp et al. (2002) Crop Sci. 42:1848-1856; Stamp et al. (2002) Maydica47:127-134). Heterosis also occurs when an existing hybrid plant(created from 2 different parent plants) is fertilized with pollen fromyet another plant, providing a subsequent boost in yield. Hybrid graincrops substantially out yield non-hybrid cultivars and also exhibitbetter response to fertilizers. However, conditions during the growingseason vary from year to year and pressures caused by environmentalchallenges, disease outbreaks and insect infestations can significantlyimpact grain yield.

Theoretically, cross pollinating hybrids can provide a yield benefit byavoiding the in-breeding depression associated with self- orsib-pollination, or by creating new gene combinations that generate aheterotic response within the kernels. This response has been termed the‘xenia effect.’ “Xenia can be defined as the effect of the pollen geneson the development of the fruit or the seeds.” (Bulant et al. ((2000)Crop Sci. 40: 182-188)

Numerous studies have shown the influence of the pollen source on thedevelopment of the kernel. Among the earliest demonstrations(Kiesselbach, T. A. (1926) Neb. Agric. Exp. Stn. Bull. 33:1-69;Kiesselbach. T. A. & W. H. Leonard (1932) J. Am. Sc. Agron. 24:517-523),Kiesselbach reported that relative to self-fertilization, crossfertilization increased kernel weights on average by 10.1% (11.8% forembryos, 10.4% for endosperm, and 3.2% for pericarp). Tsai and Tsai(Tsai, C. L. & C. Y. Tsai (1990) Crop Sci. 30: 804-808) showed anincrease in grain yield of about 30% and in increase in kernel proteincontent of about 44% in an early hybrid when it was pollinated by a latehybrid. Using maize inbred lines with normal endosperm, Bulant et al.((2000) Crop Sci. 40: 182-188) reported a relative advantage in weightof cross-fertilized to self-fertilized kernels as great as 13%. Breedingstudies at South Dakota State University confirm that cross pollinationof specific hybrids can increase kernel size and protein content, andthat cross pollination between hybrids of similar maturity accounts for40 to 60% of kernels formed in mixed stands (Wicks III, Z., (1994) Proc.Annual Corn and Sorghum Res. Conf. 4.

The development of the kernels can be altered by cross pollination(Tsai, C. L. & C. Y. Tsai (1990) Crop Sci. 30: 804-808; Poneliet, C. G.and D. B. Egli, (1983) Crop Sci. 23:872-875). Poneliet and Egli (1983)showed that the duration of the effective filling period fromcross-fertilization often was greater than that from self-fertilization.Pollen source also affects endosperm development in terms of proteincontent, amino acid profile, and translucency. (Pixley. K. V. and M. S.Bjarnason (1994) Crop Sci. 34:404-408: Bulant et al. (2000) Crop Sci.40: 182-188). At 14 DAP, the advantage of cross-fertilization on averagewas 28.8% for starch content, 24.8% for ADP-glucose-pyrophosphorylase(EC 2.7.7.27) activity, and 24.1% for neutral invertase (EC 3.2.1.26)activity (Bulant et al. (2000) Crop Sci. 40: 182-188). Tsai et al.((1991) J. Sci. Food Agric. 57: 163-174) modified P3732 endospermthrough cross-pollination, which significantly increased kernel weight,kernel protein content and grain yield across a range of fertilizer Ntreatments. The additional nutrients translocated into developingkernels of P3732 cross-pollinated plants were mainly derived fromincreases in duration of dry matter production and N uptake byvegetative tissues (Tsai et al. (1991) J. Sci Food Agric. 57: 163-174).These well-established impacts on kernel composition are the basis forthe top-cross method of producing high oil corn. The top-cross systemfor high oil corn grain production was a method used in the 1990s andearly 2000s in which high oil was induced by planting a blend of a malesterile hybrid (˜93%) and a male fertile high oil pollinator germplasm(7%). The result was an increase in oil from about 3-4% for normalcommodity grain, to about 6% for the high oil top-cross grain. The highoil grain brought a premium price per bushel at the grain elevator.(Thomison. P. R. et al. (2002) Agron. J. 94: 290-299.)

The extent of the xenia effect varies with the male and female genotype.The greater the genetic difference between the male pollen source andfemale, the greater the expected response to cross-pollination. (Leng,E. R., (1949) Agron. J. 41:555-558; Bulant, C. and A. Gallais, (1998)Crop Sci. 38: 1517-1525). The cross fertilization advantage was less forsingle-cross hybrids than for their inbred parents, and the advantagevaries with the male. For crosses between inbreds, the advantage ofcross fertilization was 13.8 and 14.5%, but only 2.5% for crosses madewith their hybrid (Bulant. C. and A. Gallais, (1998) Crop Sci. 38:1517-1525). Both pollen and maternal effects impact the response tocross pollination (Seka, D and I. Z. Cross (1995) Crop Sci. 35: 80-85;Seka. D. et al. (1995) Crop Sci. 35: 74-79).

Results of cross pollinations between hybrids observed by Bulant andGallais (Bulant, C. and A. Gallais, (1998) Crop Sci. 38: 1517-1525)illustrate that cross fertilization can increase the sink strength ofthe whole ear and that the kernel mass benefit can be observed underunfavorable conditions. The positive xenia effects have been interpretedin terms of source-sink relationships. If the resources are limiting,the increase in sink strength leads to a greater average kernel weightwith mixed fertilization than with pure self-fertilization. There was norelationship between the cross-fertilization advantage and the averageseed weight of the self-fertilized female or male pollen source.Cross-fertilization advantage was beneficial for small kernels as wellas for large kernels (Bulant, C. and A. Gallais, (1998) Crop Sci. 38:1517-1525).

Pollination success is critical to grain yield. Grain yield is measuredas the weight of grain per area of land measured at a given moisturecontent (for example, 15.5% moisture for corn). Low pollination ratesresult in poor grain yield. For this reason, grain producers typicallyrely upon self-pollination and pollination by neighboring plants in thefield since they know that the pollination will occur during the correctwindow of time because the female components of the plant will be readyto receive the pollen. Unfortunately, self-pollination will notnecessarily maximize grain yield and it is unable to account forchanging conditions and stresses that may affect the plant during thegrowing season.

Accordingly, there is a need in the industry for an invention whichallows for the improvement of grain yield, grain content, grain purity,grain characteristics, decreased contamination, or a combination ofthese attributes. The instant invention provides a method for theimprovement of grain yields by intentionally cross-pollinating theplants producing the grain. In addition, the instant invention providesa method for the increase of grain size and the modification of grainconstituents by means of specific cross-pollination with pollen from adifferent genetic background, the method for which also allows forreal-time production decisions to address conditions at the time ofpollination or to address production challenges. This method can alsoreduce undesirable contamination in the grain harvested from the field.The instant invention also provides a method of maximizing synchronouspollination with self- or sib-pollen, which provides for the reductionof contamination caused by undesirable cross-pollination, which isparticularly applicable to the organic farming industry. These twomethods can also be combined to both increase yield and modify grainsize and constituents in the same grain field, while also reducingcontamination.

The invention described herein would enable a 5% grain yield increase incorn (a conservative estimate), the annual value of this invention with33% adoption, mostly on higher productivity land, would be $1.1 billionto the entire value chain in the U.S. alone. This is based on 2015 UScorn production of 345 metric tons (U.S. Corn Growers Association (2016)World of Corn [online, retrieved on 2016 Jun. 13], Retrieved from theinternet) and a corn commodity price of $185.04 per metric ton (Jun. 13,2016 corn price [online, retrieved on 2016 Jun. 13] Retrieved from theinternet) (Calculation: $185.04/MT×345 MM MT×5% yield increase×33%adoption). Likewise, the invention described herein could enable a 5%grain yield increase in rice, which would have an annual value, with 10%adoption, mostly by larger farmers on highly productive land, of $1.5billion globally. This is based on 2013 global rice production of 746 MMmetric tons (GeoHive (2016) [online] World: Rice Production in MetricTonnes, retrieved on 2016 Jun. 20. Retrieved from the internet) and arice commodity price of $408.91 per metric ton (May 2016 rough ricecommodity price [online], retrieved from the internet on 2016 Jun. 23.Retrieved from the internet) (Calculation: $408.91/MT×746 MM MT×5% yieldincrease×10% adoption).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: This figure is a photograph of an ear of corn harvested fromhybrid H2 and pollinated with pollen from pollen donor P2 as describedin Example 2. The photograph is shown in black and white. The colorversion of the photograph shows the light colored kernels in yellow andthe darker colored kernels in purple. This ear shows the successfulcross-pollinations that occurred throughout the ear. The selfpollinations are the light kernels and the cross-pollinations are thedark kernels. This ear was useful for determining yield differencesbetween the two pollination types.

FIG. 2: This figure is a photograph of an ear of corn harvested fromhybrid H2 and pollinated with pollen from pollen donor P2 as describedin Example 2. The photograph is shown in black and white. The colorversion of the photograph shows the light colored kernels in yellow andthe darker colored kernels in purple. This ear shows the successfulcross-pollinations that occurred throughout the ear, and shows morecross-pollination than the ear in FIG. 1. The self-pollinations are thelight kernels and the cross-pollinations are the dark kernels. This earwas useful for determining yield differences between the two pollinationtypes.

FIG. 3: This figure is a photograph of an ear of corn harvested fromhybrid H2 and pollinated with pollen from pollen donor P2 as describedin Example 2. The photograph is shown in black and white. The colorversion of the photograph shows the light colored kernels in yellow andthe darker colored kernels in purple. This ear shows very high levels ofcross-pollination (greater than 85%). The self-pollinations are thelight kernels and the cross-pollinations are the dark kernels.

SUMMARY OF THE INVENTION

Provided is a method of improving commercial grain production of anyplant species by intentionally cross-pollinating grain plants withpollen from a different genetic background. This method provides a yieldincrease as a result of heterosis, or the xenia effect. This method hasthe additional benefit of providing for modification of graincharacteristics, including grain content and grain constituents.

Also provided is a method of enhancing levels of self- and/orsib-pollination in commercial grain production, the method including theintentional application of designated male pollen (with the same geneticbackground as the designated female plant) to a designated female planton one or more occasions. At the time of the intentional application,the designated female plant is in a state when the female components areready to receive pollen. The male components of the designated femaleplant may or may not be releasing pollen at the time of the intentionalapplication of designated male pollen. In the event that the malecomponents are releasing pollen, some female components will receive theintentionally applied designated male pollen while other components mayreceive self-pollen or sib-pollen from neighboring plants, resulting inan overall increase in successful pollination events, and therebyreducing contamination caused by undesirable outcrossings.

One embodiment of the invention is a method of increasing grain size andmodifying grain characteristics, including grain content andconstituents, by means of specific cross-pollination. For example, theoil, protein or starch content of the grain may be modified, and thespecific oil, protein or starch constituents of the grain may also bemodified. This method provides for the use of real-time productiondecisions to address specific conditions at the time of pollination, andallows the grower to address specific production challenges in thefield. At the time of the intentional application, the designated femaleplant is in a state when the female components are ready to receivepollen. The male components of the designated female plant may or maynot be releasing pollen at the time of the intentional application ofdesignated male pollen. In the event that the male components arereleasing pollen, some female components will receive the intentionallyapplied designated male pollen while other components may receiveself-pollen or sib-pollen from neighboring plants. In the event that themale components are not releasing pollen, the majority of the femalecomponents will receive the intentionally applied designated male pollenfrom a different genetic background, resulting in heterosis, or thexenia effect, and a concurrent increase in the size of grains orkernels, as well as intentional changes in grain content orconstituents.

Both these methods can also help to reduce contamination in the graincaused by undesirable pollination events. The methods may also becombined in order to achieve both a grain yield increase and an increasein grain size, along with the modification of grain characteristics.

The methods are applicable to a wide range of plants including, but notlimited to, corn, soybean, wheat, rice, sunflower, canola, sorghum,cotton, barley, and pearl millet. The application of the designated malepollen may be conducted by automated means, mechanical means, positivepressure means, negative pressure means, manual means, semi-automatedmeans, or combinations thereof: Moreover, the designated male pollensource may be from previously preserved or freshly harvested pollen frommale source material. The pollen may have been harvested from one ormore of a field, a growth chamber, a greenhouse, a glasshouse, a shadehouse, a hoop house, a vertical farming facility or a hydroponicfacility. Preserved pollen may have been preserved by any means thatpermits the pollen to retain viability, including but not limited tovarious forms of cooling or freezing including, but not limited to,chilling, cryopreservation, freeze drying, or storage in liquidnitrogen. Furthermore, the pollen, whether fresh or preserved, may havebeen collected from a source with altered circadian rhythm or a sourcewith normal circadian flowering, but wherein said male components ofsaid designated female parent plants are delayed, or allowed to shedwith no delay. The pollen, whether fresh or preserved, may have beencollected from one or more sources and may have been combined withpollen from other sources before application.

Further provided is a method of improving crop productivity. The methodincludes intentionally applying specifically selected pollen from agenetically unrelated source and allows the opportunity for makingreal-time, agronomic-specific decisions in selecting the unrelatedpollen sources. This method allows the maximization of seedcharacteristics for any given environmental condition or market needsfor any given plant of interest. For example, in one embodiment, thespecifically selected pollen is obtained from sources optimal forapplication based on environmental conditions, including abiotic andbiotic conditions. In another embodiment, the specifically selectedpollen is obtained from a source optimal for application based on plantperformance data (such as data obtained from yield trials, for example),or commodity and market price information. In yet another embodiment,the specifically selected pollen influences the characteristics of thegrain harvested from the female plant, impacting characteristics such asgrain content and constituents, which include, but are not limited to,grain oil content and composition, grain starch content and composition,and grain protein content and composition.

In an embodiment of this invention, the designated female plants may beeither male sterile or male fertile, and the pollen source may beapplied prior to exposure from a designated female plant pollen,including self-pollen or it may be applied concurrently while the plantis receptive to self-pollen and/or sib-pollen. Moreover, the method mayinclude male-specific pollen sources which are optimal for applicationof pollen based on environmental conditions or on market trends whichwould make seeds with a specific composition (i.e., starch, oil, droughtresistance, flood tolerance, etc.), more valuable. Furthermore, thepollen may be selected from a single source or a combination of sourcesin order to enable a combination of traits from seed to seed.

DETAILED DESCRIPTION

The following is a detailed description of an embodiment of technologyand methods enabling improved grain yield and modification of grainsize, content and constituents. Such technology and methods may be usedin association with any plants for which it is desired to produce grain.For ease of discussion and understanding, the following detaileddescription often refers to the invention for use with maize (alsoreferred to as corn). It should be appreciated that the technology andmethods may be used with any plants, and corn or other specificallynamed plants are discussed for illustration purposes only and are notintended to be limiting. The technology may be used in conjunction withany grain plant including those that are hybrids, non-hybrids, malefertile and male sterile.

Grain is produced for a number of purposes, including human consumption,animal consumption, industrial use, re-planting, and for researchpurposes. The primary goal of grain production is the harvesting of ahigh-yielding, high quality grain. Seed companies devote billions ofdollars to research in the pursuit of developing better plant geneticsin order to improve grain yields. Regardless of the end use of thegrain, the production of grain is dependent on the appropriate malepollen fertilizing the appropriate female plant at the appropriate time.

The synchronization between the male pollen and the female receptivityis referred to herein as reproductive synchrony. Reproductive synchronyis easily achieved through self-pollination or pollination byneighboring plants of the same variety. However, reproductive synchronyis more difficult between different varieties which may mature atdifferent times or as a result of different temperatures, as well asother environmental factors. A grain producer might have an interest inpollinating a crop with pollen from a variety that could impart specificcharacteristics to the resulting grain. Typically, this cannot beachieved unless the following circumstances exist: (1) the producerknows at the beginning of the growing season which variety they wish topollinate with; (2) the varieties will be releasing pollen and receptiveto pollen at the same time; and (3) the varieties are grown in closephysical proximity to each other. So, for example, if a producer isexperiencing a period of drought and would like to pollinate with avariety that shows strong grain yields even during drought periods, hewill be unable to do so unless that is the variety that he has alreadychosen to plant and grow and it is already in his field, as well asreleasing pollen at the appropriate time. This producer cannot typicallypollinate a crop with pollen from a variety that might help overcomepotential low grain yields due to drought if the variety is being grownelsewhere and has a different maturity period in comparison to hisalready planted variety. The present invention is able to overcome allthree of these potential barriers to reproductive synchrony, therebyenabling a broader choice of pollen for the producer as well as theability to make real-time production decisions.

Synchronous pollination within and between corn ears is particularlyimportant in maize because it has been shown to improve kernel set,which is a critical factor in grain yields. Carcova (Carcova et al.(2000) Crop Sci. 40: 1056-1061) demonstrated that synchronouspollination of exposed silks on apical and sub-apical ears 5 days aftersilking showed an improvement in kernel number per plant.

Close synchrony between male and female flower anthesis, and a minimumpollen density per exposed silk are fundamental requirements for highlevels of seed production and genetic purity. Previously collected andpreserved pollen, or fresh pollen, can be applied to receptive silksover a period of seven to ten days, ensuring seed set under conditionsof decreased pollen viability, silking delay relative to pollen shed, orpoor ‘nick’ between male and female inbreds. In the agronomic industry,“nick” is a term used in hybrid seed production that refers to thesynchrony of both male and female flower types (i.e. the peak of pollenshed by the male aligned with the peak of silk emergence in the female)developing at the ideal time such that pollen grains will successfullypollinate, resulting in fertilization. For grain producers, the perfect“nick” occurs when 50% of the male population begins to shed pollen onthe same day when 50% of the female population begins to exert silks.

A real-time production decision is a decision that is made during thependency of the growth and maturation of the crop, rather than adecision that is made during the planning stage prior to the planting ofthe crop. Grain producers choose which grain variety to plant in anygiven production season based on characteristics that are optimal fortheir climate, land, growing conditions, and other factors. Once thatdecision has been made, and the crop has been planted, growers makereal-time decisions during the growth of the crop, such as when to sprayfor weed control or treatment of disease or insect problems. Thesedecisions occur as the crop grows in response to specific conditions andstresses that the crop is facing at any given time. Prior to thisinvention, however, grain producers have not been able choose pollen asa real-time production decision because the pollen used in grainproduction comes from the plants already growing in the field. Thus, thepollen choice is typically made at the time that the grain variety waschosen, prior to any planting, and without any knowledge of specificconditions that may occur later in the growing season affecting grainyield and viability.

The present invention is an improved method of grain production thatincludes collecting, storing, and delivering pollen from male plants tothe female plants. Some methods of collecting pollen are known in theart. For example, U.S. Pat. No. 4,922,651 discloses an apparatus foreffecting or improving pollination of plants.

In addition, some methods of pollen delivery, such as U.S. Pat. No.4,922,651, are known in the art. In addition, various methods of pollenstorage are known. For example, U.S. Pat. No. 5,596,838, covering amethod and instrument for the preparation of pollen for cryogenicstorage, teaches that pollen can be stored for periods of months andstill remain viable. Although some methods of pollen delivery are known,these methods do not teach, suggest, or motivate the user tointentionally deliver pollen in such a way that allows the grower totake advantage of heterosis while also enabling real-timedecision-making to improve yields based on current conditions. Thepresent invention results in pollinations which occur prior to theactive period in which the plant might be subject to self-pollinationand/or be exposed to other undesirable pollination, or during the activeperiod in which the plant is subject to self-pollination and/or exposedto other pollination, thereby increasing pollination efficiency. Thepresent invention increases the heterosis effect, and allows forreal-time production decisions. The invention does not require the useof male sterility or isolation, although one or both may be employedwithout departing from the scope of the invention.

As discussed above, the current invention is applicable to the commonpractice of planting seed that will germinate and become seed bearingplants that will bear the grain to eventually be sold to grain elevatorsor other customers, used as animal feed, or used in research programs.However, rather than relying exclusively upon self-pollination orpollination from neighboring plants as is the case with currentpractices, this invention provides an improved alternative by increasingsuccessful self- and/or sib-pollination, and/or by modifying the levelof cross-pollination through the intentional application of male pollento female plants at a specific time. Use of the term “intentional” withregard to pollen application means the specific application of pollen ina way that does not include natural pollination by wind, insect activityor other naturally-occurring conditions. Intentionally applied pollen ispollen that has been applied to a plant as a result of a deliberatehuman activity or decision, and may be applied by hand or by othermeans. One example of intentional pollination is the “prescriptive”application of pollen, which is the use of pollen to solve a problem inorder to address a specific need or condition in the field. The specificpollen to be used and the timing of the intentional application willdepend upon the need or condition being addressed.

The acquisition of male pollen [sometimes “pollen^(M)” ] required tomake seeds that will mature into grain can be via a pollen bank. Apollen bank is a source of stored pollen that has been collected fromone or more pollen sources and stored in such a way that the pollenretains its viability. The plants that have been used as the pollensource for such a pollen bank may have been grown and harvested in anyconditions, including but not limited to, a field, a growth chamber, agreenhouse, a glasshouse, a shade house, a hoop house, a verticalfarming facility or a hydroponic facility. Pollen from a pollen bank mayhave been sourced in different ways. For example, in one embodiment,fresh pollen can be harvested from males grown in a controlledenvironment in which the circadian rhythm is 2-8 hours ahead ofnaturally growing female plants in the field. This method will befurther detailed below. In another embodiment, the pollen which isstored in the bank may be preserved pollen that was collected days,weeks, months or years prior to its eventual removal from the bank forpollinating purposes. Preserved pollen may have been preserved by anymeans that permits the pollen to retain viability, including but notlimited to various forms of cooling or freezing including, but notlimited to, chilling, cryopreservation, freeze drying, or storage inliquid nitrogen.

In one or more embodiments, the pollen may be harvested from an antherstudio. The anther studio enables optimal growth conditions for maleplant reproductive tissues for any species or variety of plant. Thetissues (corn tassels for example) are cut from plants growing instandard outdoor conditions, such as in the field or those grown incontrolled conditions, such as the greenhouse or a growth chamber. Thetissues are preferably cut prior to the plant beginning to shed pollenand are placed into the anther studio. The tissue may then be culturedin a nutrient medium allowing for further growth. At least one ofspecialized lighting, temperature, and/or humidity may be cycled in theanther studio, allowing for continued growth of the tissue. Growth maybe modulated to increase or slow the rate of growth and thus modulatethe duration for availability of pollen. This enables the ability tohave on demand pollen for pollinations that can be accomplished at anytime of the day or night. This has utility for pollination enablement ofseveral beneficial and valuable processes related to seed and grainproduction. It also provides concentrated sources of pollen forpreservation purposes. Any pollen harvested from the anther studio andpreserved could be utilized in the same manner as the freshly harvestedpollen, but at a duration long after the fresh pollen, which has notbeen preserved, has died. (R. I. Greyson (1994) Maize inflorescenceculture. p. 712-714. In: M. Freeling, V. Walbot (eds), The MaizeHandbook; Springer-Verlag, New York: J. B. Schoper, R. J. Lamber, B. L.Vasilas, and M. E. Westgate (1987) Plant factors controlling seed set inmaize. The influence of silk pollen, and ear-learf water status andtassel heat treatment at pollination, Plant Phyiol. 83: 121-125)

Mechanical delivery of the pollen^(M) can occur as soon as females arereceptive, which is always prior to the designated female pollen[sometimes pollen^(F)] becoming viable on any given day, thus enabling asuccessful cross pollination with all receptive females. In other words,the female component of the plant is open to receive pollen from a malebefore the male component of the same plant is actually ready to producepollen. In corn, females are receptive to pollen when the silks areexposed to receive the pollen. The silks are receptive to pollen priorto emergence and remain receptive for many days after emerging from thehusks. Moreover, in corn, two possibilities exist: pollen may be shedprior to silk emergence (protandry), or silk emergence may be prior topollen shed (protogyny). In either case, the silks will be receptive topollen before pollen is shed on a given day. This invention isapplicable to both situations. Moreover, in some examples, the silks arereceptive to pollen all day for about seven days. Accordingly, pollenmay be intentionally applied any number of times, including but notlimited to, once per day, twice per day, or in a continuous application.Alternatively, mechanical delivery of the pollen^(M) can occur whilefemales are viable and also during the period in which the designatedfemale pollen [sometimes pollen^(F)] becomes viable on any given day,thus enabling both self-pollination with pollen^(F) simultaneously withintentional pollination by pollen^(M). Without the use of malesterility, in corn, for example, pollen^(F) typically will begin to shedin the mid-morning hours, and continue to late morning or earlyafternoon, after which all pollen^(F) either finds a viable female andgerminates, enabling a successful self-pollination, or dies within fourhours or less if it does not land on a viable female (Luna V. et al.(2001) Crop Sci. 41(5): 1551-1557). Thus, the female corn plant has adaily window of time during which male pollen can be delivered andduring which pollen^(F) is not shedding. This window will be repeatedover several days. The current invention cross allows for thepollination of nearly all of the viable females during a window of timeduring which pollen^(F) is not shedding or is not viable, or acombination of cross-pollination and self-pollination. The currentinvention has the capacity to cross pollinate nearly all of the viablefemales prior to pollen^(F) becoming viable or after pollen^(F) becomesviable, depending on the preference of the grain producer and thesituation in the field.

Accordingly, one aspect of the invention is the timing of pollendelivery to female plants. In all crops, there is a daily cycle duringthe pollination window (the time during which the female is receptive topollen and during which a successful pollination event can occur) inwhich females mature and grow relatively continuously, while males havea distinct cycle in which pollen becomes viable and sheds or is dehiscedfrom the anthers beginning in mid-morning and ending in late morning orearly afternoon. In some cases, pollen^(F) can become viable prior tothe females being fertile, but there will always be a first morning uponwhich viable females become fertile prior to viable pollen^(F) beginningto shed on that day. Therefore, pollen^(F) that may have shed theprevious day has long since died and become inviable prior to femalesbecoming viable the next morning. The early to mid-morning hours aretherefore ideal for application of pollen^(M) to fortuitously crosspollinate all viable females just prior to pollen^(F) beginning to shed,although one may choose to apply pollen several days before pollen^(F)beginning to shed or at several times of the day or in a continuousfashion even during the shed of pollen to increase the probability ofsuccessfully increasing heterosis effects, and maximizing the yield ofgrain. In one example, pollen may be delivered at 6:00 a.m. However,delivery of pollen may occur at any hour of the day, providing a greatadvantage over traditional methods.

By delivering pollen at the appropriate time and for the proper durationof a plant's fertility window, adequate pollination can be achieved.This invention enables for the first time effective grain productionwithout solely relying upon self-pollination or pollination byneighboring plants which may, at the time of pollination, be anundesirable pollination option due to any number of biotic or abioticstresses upon the crop. Undesirable biologically compatible sources canbe referred to as pollen^(U). The present invention allows fordesignated pollination such that when pollen^(U) begins to shed, allviable target females have already been pollinated by the selectedpollen source and the pollen^(U) dies after a short period of timedepending on species and environmental conditions (Luna V. et al. (2001)Crop Sci. 41(5): 1551-1557; Stanley, R. G. & Linskens, H. F. (1974)Pollen: Biology, Biochemistry, Management. Springer-Verlag. Heidelberg.;Shivanna, K. R. (2003) Pollen Biology and Biotechnology. SciencePublishers, Inc.) without finding any viable target females, thusavoiding the pollination of females by an undesirable pollen source.This would be applicable to situations in which self pollen orneighboring plant pollen would be considered to be pollen^(U). Differentspecies of pollen have different lifespans, which may be furtheraffected by environmental conditions (Dafni, A. & D. Firmage (2000)Plant Systemics and Evolution 222(1): 113-132). Higher humidity andlower temperatures may extend pollen longevity. For example, in rice(Oryza sativa), pollen longevity has been found to be as short as 4minutes (Koga et al. (1971) Cytologia 36: 104-110) or up to 20 minutesfor 50% of the pollen to die (Khatum, S. and T. J. Flowers (1995) J.Exp. Bot. 46: 151-154). In contrast, field grown radish (Raphanussativas) pollen was shown to have a 5-day lifespan (Siddiqui, B. A.(1983) Acta Bot. Ind. 11: 150-154).

The capacity to deliver viable pollen on demand to effect timelypollinations of receptive silks addresses a number of limitations commonto grain production on a field scale. Specifically, prescriptive use ofthe invention overcomes low levels of pollen production and eliminatesproblems caused by poor reproductive synchrony between male and femaleflowers.

Pollen^(M) can be delivered in any number of ways, including, but notlimited to, manual delivery, manual delivery with a small handmechanical device for semi-automated dispersal, by field drivenmachinery containing pollen dispersal machinery or via fully automateddispersal by a self-propelled and/or human guided apparatus such as adrone that has a pollen dispersal device mounted to it, wherein thepollen dispersal is by automatic or semi-automatic means, including, butnot limited to, positive pressure, negative pressure, mechanical orpneumatic means. Use of a drone would be especially novel and practicalin this method. Small drones, which need not be regulated, can be usedin the method, and can be guided using GPS coordinates to focus thepollen dispersal directly over the female plants. Using any of thesemethods, it has been estimated that about 140 grams (5 ounces) ofpollen^(M) fortuitously delivered for 3-4 consecutive days is enough tosuccessfully cross pollinate one hectare (2.47 acres) of maize females.This estimate is based on literature that provides the number of maizepollen grains per mg, and estimating approximately 4-5 grains or pollenper silk, or fewer, for successful pollination (3-4 grains of pollen persilk is commonly accepted by those skilled in the art of pollination asthe amount needed to ensure successful pollination (Westgate, M. E. etal. (2003) Crop Sci. 43: 934-942)). See, for example, Porter (1981)Environ. Health Perspectives 37: 53-59; Miller (1982) In Maize forBiological Research. W. F. Sheridan (ed.), pp. 279-293). The timing ofpollen delivery, amount of pollen required, and number of days of pollendelivery can be adjusted as necessary for circumstances such as croptype and weather patterns, (i.e. crop height, crop density, rainfrequency or amount, wind speed and direction, etc.). For example, themethod can be adjusted for soybeans which have a longer pollinationperiod than maize. Moreover, it is anticipated that the efficiency ofthese delivery methods will reduce the amount of pollen needed forsuccessful pollination. It is estimated by the inventors that these moreefficient methods may use 1/1000 of the amount of pollen currentlyproduced for grain production. The inventors' research indicates thatperfect corn kernel set requires about 3000 pollen grains per silk asshed normally in the field from maize tassels. Hand pollination studiesand open field studies indicated about 3 to 4 pollen grains per silkensure kernel set (M. E. Westgate et al. (2003) Crop Sci. 43: 934-942;M. Uribelarrea et al. (2002) Crop Sci. 42: 1910-1918). This results in aratio of about 1/1000^(th) of the amount of pollen currently requiredfor grain production.

One skilled in the art can easily vary from the examples listed above asnecessary. For example, in some situations, it may be advantageous topollinate at night. In others, it may be advantageous to pollinate allday. In maize, pollination efficiency is improved when silk health isoptimal and silk viability is at its maximum, which often coincides withconditions when the temperature is not as hot and when there is highmoisture content in the air, meaning the silks are less likely to dryout quickly. Thus, a corn producer may choose timing of pollination tooccur at the point when silk health and viability is optimal.

This invention can operate in any crop plant to either improve or enablegrain production. It can operate in any environment including, but notlimited to, ideal or target growing environments, off-seasonenvironments, or controlled environments (e.g. shade/glass/green/hoophouses, growth chambers, vertical farming facilities, hydroponicfacilities, aeroponic facilities etc.). Pollen^(M) can be applied assoon as females are viable and irrespective of pollen^(F) or otherundesired pollen viability. This invention can also be used to improvecurrent grain production practices even when all conventional componentsare present, thus improving grain yields in challenging productionsituations.

In one embodiment, the invention may be used to solve or reduce the losscaused by drought. This is advantageous for all in the industry,including producers and crop insurers. Specifically, in a drought,plants become stressed. When a plant is stressed it often produces malecomponents only. Accordingly, the pollen is there, but there are nofemales to receive it. Sometimes the plant will produce the femalecomponents later, but at that point the pollen is no longer availablefor pollination. By employing the method of the present invention,pollen may be delivered from a pollen storage bank or controlledenvironment to the stressed plants after the females are available forpollination, thus rescuing the plants to at least some degree to produceharvestable yields of grain.

Moreover, in another embodiment of the invention, real-time research orproduction decisions can be made with regard to the pollen intended tobe used for pollination, such as the desire to produce grain withspecific characteristics desired by the producer, including, but notlimited to, increased oil production, decreased oil production,increased starch production, decreased starch production, improveddrought resistance, improved resistance to flooding or other sources ofexcessive moisture, improved performance in high heat conditions,improved performance in low heat conditions, improved fungal resistance,improved insect resistance, increased yield, and improved pollinationwhen the initial intended pollination is out of synchrony due to themale and female reproductive cycles being out of synchrony. All of thesesituations may be addressed by selecting pollen sources specificallyoptimized to maximize yields for any given condition for any givengrain-producing crop of interest. Having a variety of pollen sourcesstored within a pollen storage bank enables these type of research orproduction decisions to be made just prior to pollen application.Specifically, the designated male pollen may be applied prior to, orduring, self-pollination or exposure from the viable pollen fromneighboring plants. The pollen storage bank from which the grower issourcing their pollen may include male specific pollen sources optimalfor application of pollen based on environmental conditions. Moreover,any pollen used in the present invention, including this embodiment, mayinclude pollen from a single source or from multiple sources pooledtogether.

An example of a real time production decision includes the use ofperformance data in order to decide which male to use in a given hybridcross or in grain production. For example, winter grain production isoften practiced in the southern hemisphere outside of northernhemisphere growing seasons. Often, grain fields are planted in thesouthern hemisphere prior to complete performance data being collectedin the northern hemisphere. Incomplete and/or delayed planting decisionsare often made, resulting in poor decisions on the choice or volume ofplants to be planted on the limited available land in the southernhemisphere. The current invention allows the producer to plant thefemale and then wait approximately 45 to 75 extra days in order to makea more informed decision about which male to use for pollinating femaleplants. This type of production decision would also apply for grainproduction in which a northern grain producer is waiting for morecomplete data from the southern hemisphere in order to decide which maleto use.

As will be appreciated by one of skill in the art, the selection of themale pollen to be used in the practice of the invention is a significantfactor in the potential benefit recognized by practicing the invention.Pollen vigor and viability are considerations, along with the varioustraits and characteristics that can be conferred by the pollen. Theremay be circumstances when using a pollen with lower vigor or viabilityis still a good choice because of the traits that it will confer uponthe resulting grain. In such circumstances, when a pollen is known tohave a lower vigor or viability, a more moderate resulting yieldincrease should be expected. Such decisions can be made by the growerbased on the particular characteristics of the crop, the desiredresulting grain, and prevailing conditions at the anticipated time ofpollination with regard to environment and other biotic and/or abioticpressures affecting the crop. Accordingly, the ability to select thepollen at the time of pollination is a further significant factor in thepotential benefit recognized by practicing the invention. Table 1,below, outlines selected potential benefits of using the invention.

TABLE 1 Example Benefits of Pollen Selection Self Pollina- tion, CrossProduction Application of the Pollina- Problem Invention ExpectedBenefit tion or Both Yield is limited Use of fresh or Yield increaseCross due to self or sib preserved pollen pollination in with adifferent grain production genetic background applied to receptive silkson demand Poor pollen Use of fresh or Yield increases Both production bypreserved pollen resulting from males, not applied to receptive improvedsuitable for silks on demand pollination grain production therebyincreasing on a field scale pollination Poor reproductive Use of freshor Increased grain Both synchrony preserved pollen yield per acre,between male applied to receptive decreased self and female silks ondemand pollination leading to low thereby increasing of females, grainyield a pollination decreased out- crossing from adventitious sourcesLow yield in Use of fresh or Increased yield of Both top-cross preservedpollen high value grain production of applied to receptive ensured byhigh value grain silks on demand directing required thereby increasingmale pollen to pollination receptive silks, and potential foreliminating male pollinators from the grain field Organic or Use offresh or Increased purity Self non-GMO preserved pollen of organic orpollination contamination applied to receptive non-GMO or silks ondemand specialty grain thereby increasing purity Oil production Use offresh or Increased yield Cross modification preserved pollen andincreased or pollination desired containing traits decreased oil forincreased or output in grain decreases oil production applied toreceptive silks thereby changing oil output Starch Use of fresh orIncreased yield Cross production preserved pollen and increased orpollination modification containing traits decreased desired forincreased or starch output decreased oil in grain production applied toreceptive silks thereby changing starch output Drought Use of fresh orRescue from Cross conditions preserved pollen drought. pollination priorto to hit the nick Yield increase pollination wherein the pollen may ormay not contain drought resistance traits Protein Use of fresh orIncreased yield Cross production preserved pollen and increased orpollination modification containing traits for decreased desiredincreased or protein output decreased protein in grain productionapplied to receptive silks thereby changing starch output Excessive Useof fresh or Rescue from Cross heat or cold preserved pollen extreme heatpollination conditions containing heat or or cold. Yield cold resistanceincrease traits Pressure Use of fresh or Rescue from Cross from funguspreserved pollen fungal pressure. Pollination containing fungal Yieldincrease resistance traits Pressure Use of fresh or Rescue from Crossfrom insects preserved pollen insect pressure. Pollination containinginsect Yield increase resistance traits

As will be appreciated by one of skill in the art, the technology ofthis invention can be used to modify cross-pollination by increasing thelevel of cross-pollination (for example, in order to increaseheterosis/xenia effect and thereby increase yield of a maize grainproduction field) but also can be used to reduce the level ofcross-pollination and instead focus on self-pollination (for example, inan organic or non-GMO grain production scenario in order to increasesuccessful self-pollination events and thereby reduce undesirablecross-pollination events). In such a case, the instant invention can beused to specifically avoid cross-pollination by enhancingself-pollination and thereby ensuring lower levels of crosscontamination from undesirable pollen from GMO sources.

In addition, the current invention allows the producer to use a varietyto pollinate the crop which would not normally be able to reach maturityor be a suitable pollinator in the producer's climate. Such a varietymight have excellent characteristics that would make it a suitablecross-pollinator in order to maximize grain yield, but it cannot begrown in the producer's climate or will not be able to reach the stageat which it releases pollen in the producer's climate. The presentinvention overcomes this limitation and allows the producer a broaderrange of pollen types from which to select the ideal candidate for theparticular crop and any particular stresses that may be present in thecrop as the time for pollination approaches.

Another real time production decision made possible by the currentinvention is the decision of which pollen to use based on market andcommodity price information. A grain producer would have approximately45 to 75 extra days to watch the markets and decide which male to usefor a specialty grain market, such as waxy corn versus food grade cornversus #2 yellow dent (commodity grain market).

Real time production decisions could also be made using environmentaldata. For example, a grain producer may have experienced a drypre-flowering period and therefore may choose a male to pollinate thefemale that is known to be drought resistant. This decision will reduceseed abortion during drought stress, thus increasing yields incomparison to allowing the hybrid to self-pollinate to produce grain. Asimilar situation would exist for a producer experiencing high pressurefor seed-feeding pests. A male could be selected that includes a genefor resistance to seed-feeding pests which will protect the developingseed and thus increase the hybrid seed yield relative to the yields thatwould have been obtained by using a male that was originally planned tobe used before the pest problem arose, or that would have been plantedin the more conventional block system. Environmental data used in realtime production decisions could include, but is not limited to, abioticconditions such as drought, nitrogen or other nutrient availability,humidity variation, and extreme temperature conditions; as well asbiotic environmental conditions including, but not limited to, insectand nematode pest pressure, weed pressure, and disease pressure.

The following examples illustrate the present invention in more detailand are illustrative of how the invention described herein could beimplemented in corn. The basic method could apply to any crop with cropspecific modifications as appropriate. Examples 1 and 2 occurred in corngrain production fields in central Iowa during the summer of 2015.

Example 1

The invention described herein was practiced using a commercial hybrid(H1) as a female in one location in central Iowa. Blocks of 12 rows(width of 0.76 m (2.5 feet) per row for a total of 9.1 m (30 feet) inwidth) that were 13.7 m (45 feet) long of each hybrid were grown. Onepollen donor inbred (P1) that was unrelated to H1 was grown at adistance of greater than 45.7 m (150 feet) away from the hybrid block.The pollen donor was a publicly available line from the USDA-ARSNational Plant Germplasm System, and carried a gene for expression ofanthocyanin in the endosperm of the kernel. When P1 is used as a malepollen source to pollinate, successful pollination of kernels is quicklyobvious to the naked eye by observing the purple color of the maturekernels on an ear.

To practice the invention, water was sprayed on the tassels of the H1grain production block (H1) at about 8:15 am to delay pollen shed fromthese female rows until about 8:45 am. After spraying the water on thefemale tassels, the invention was immediately practiced by coercingpollen^(M) from the tassels of P1 and collecting the pollen in bags. Thepollen^(M) was then immediately applied to the H1 silks by hand usingtechniques well known in the art involving gentle tapping on the bag ofpollen to release the pollen grains onto the silks. These pollinationsoccurred prior to the hybrids shedding any pollen. There were 13pollinations made into the H1 grain production block by directing pollenonto the silks of the plants of H1 that were in the middle of the block.This imitated a case in which pollen may have been harvested by a nearbyfield of an unrelated corn hybrid, put into storage for a short periodof time, and then brought out of storage in the early morning hoursbefore pollen begins to shed from the hybrid grain production fieldbeing used as a female. Yellow kernels that are on these ears at harvestcan be assumed to be mostly, if not all, self-pollinations from the H1plants in the grain production block, while the purple kernels on theseears are the result of cross pollinations from the P1 pollen donor. Itis important to note that manual direction of the pollen was veryprecise and measured, as there were only 2 purple kernels found on 6adjacent ears that did not have P1 pollen directly applied to them(Table 2). These ears and resulting kernels served as controls tomeasure basal self-pollination percent in the block. Therefore, it canbe inferred from this data that self-pollination frequency in the middleof this block of hybrids was very close to 100% under conventionalmethods of grain production. This experiment replicated what might beexpected with the invention where pollen may have been harvested by anearby field of an unrelated corn hybrid, put into storage for a shortperiod of time, and then brought out of storage in the early morninghours before pollen begins to shed naturally from the hybrids being usedas females

The results in Table 2 show that the invention is very effective atincreasing cross-pollination in this grain production field. Among the13 ears upon which the invention was practiced, there were a total of6,564 kernels, 2034 of which were purple, or 31% of the total. This is ahuge increase in the frequency of cross-pollination of more than 38,000%(or 38-fold) compared to the 6 ears that served as a control. It isimportant to note that P1 and the other pollen donors mentioned inExample 2 below are non-elite inbreds that are more than 20 years oldand were never bred for commodity grain yield. Further, based on theliterature (Sari Gorla, (1975) Theor. Appl. Genet. 46:289-94), hybridpollen, such as that from H1, is more fit and vigorous than inbredpollen. As a result, the P1 pollen had a competitive disadvantage invigor and fitness compared to the H1 pollen. This further illustratesthe effectiveness of the invention to increase cross-pollination.

TABLE 2 The effect of the invention on self- versus cross pollinationpercent in H1 using P1 male. On-demand Open-pollinated invention # of %of # of % of Pollination type kernels total kernels total Hybrid selfpollination 2452 100% 4530 69% (yellow kernels) Hybrid cross pollination2  0% 2034 31% (purple kernels) Total: 2454 6564 Increase in crosspollination 38021% frequency

Example 2

The invention was practiced using 2 commercial hybrids (H1 and H2) asfemales in one location in central Iowa. Blocks of 12 rows (width of0.76 m (2.5 feet) per row for a total of 9.1 m (30 feet) in width) thatwere 13.7 m (45 feet) long of each hybrid were grown. Three pollen donorinbreds (P1, P2, and P3) that were unrelated to these commercial hybridswere grown at a distance of greater than 45.7 m (150 feet) away from thehybrid blocks. The three pollen donors were publicly available linesfrom the USDA-ARS National Plant Germplasm System, and carried a genefor expression of anthocyanin in the endosperm of the kernel. When usedas a male pollen source to pollinate, successful pollination of kernelsis quickly obvious to the naked eye by observing the purple color of themature kernels on an ear.

In the very beginning of the pollination window, the invention waspracticed by coercing pollen^(M) from the tassels of the pollen donor(P1, P2, and P3) males and collecting the pollen in the bags. Thepollen^(M) was then immediately directed to the H1 and H2 silks by handusing techniques well known in the art involving gentle tapping on thebag of pollen to release the pollen grains onto the silks. H1 was earlymaturing, as was P1, so P1 was used to pollinate onto H1 silks. Itshould be noted that a portion of the H1 ears in this experiment arecomprised of 11 of the ears referred to in Example 1 above that fit thecriteria to be included in this data set, and therefore were the resultof the “spraying of water” to delay the males of H1 and werepollinations made prior to shedding of any H1 pollen. H2 was latermaturing, as were P2 and P3. Therefore P2 and P3 were used to pollinateonto H2 silks. All pollinations onto H2 and the majority of thepollinations onto H1 occurred during the early onset of the pollinationwindow in the morning, with the exception of the 11 pollinations onto H1that occurred prior to the natural pollen window that were described inExample 1 above.

More than 75 hand pollinations were made into H1 and H2 by manuallydirecting pollen onto the silks of the plants of H1 and H2 that were inthe middle of the block during the early part of the natural pollinationwindow. This method is a manifestation of the invention in which freshpollen could be harvested in the early morning natural pollinationwindow and immediately applied in a directed manner to a neighboringunrelated hybrid field. Further, this experiment imitated, although notexactly, a case where pollen may have been harvested by a nearby fieldof an unrelated corn hybrid, put into storage for a period of time, andthen brought out of storage in the early morning hours before pollenbegins to shed from the hybrids being used as females, and applied tothose females. Pollinations were labelled with regard to which of thepollen donors was used for each pollinated ear. Yellow kernels that areon these ears at harvest can be assumed to be mostly, if not all,self-pollinations, while the purple kernels on these ears are the resultof cross pollinations from the pollen donors. As noted in example 1above, it can also be inferred that selfing in the middle of the blocksof hybrids in this experiment was also very near 100% when usingconventional grain production methods.

A portion of the harvested ears from the intentional pollinations had ahigh percent of cross pollinated kernels, often above 50%. These earsoften had large blocks of purple kernels in one part of the ear, whilethe rest of the ear was yellow. This phenomenon can be seen in FIG. 3,which shows an ear with approximately 85% cross-pollination as shown bythe high number of dark (purple) kernels. In other cases, there wereears that had a lower percent of purple kernels, often in the 10-30%range. Among these, there were a total of 49 ears that were asegregating mix of purple and yellow kernels, rather than solid blocks.Two sample ears are shown in FIGS. 1 and 2. These ears show a clear mixof dark (purple) and light (yellow) kernels. These ears wereparticularly diagnostic to illustrate the yield effect of self vscross-pollination, whereas the ear shown in FIG. 3 was not diagnosticfor illustrating yield because there were not enough self-pollinationsto establish a sample size large enough for the comparison. The kernelson these ears developed in nearly identical environments, with the onlydifference among them being that they had different genetics from themale parent (self-pollination vs. cross). These kernels were harvestedand separated into two groups for each ear: 100 yellow and 100 purplekernels from approximately the middle of the ear (sometimes when therewere not enough purple kernels, a lower amount may have been used, witha minimum of 40 kernels of each type). Measurements were taken on thesesets of kernels for each ear. Table 3a and 3b show the differencesbetween yellow vs purple kernels from these ears for weight per kerneland test weight (weight per volume). The data from Tables 3a and 3billustrate several points:

1. Practicing the invention resulted in higher yield for at least onemeasure (kernel weight or test weight), for the cross pollinated kernelsthan self-pollinated kernels in all three crosses (Tables 3a and 3b).

2. Practicing the invention may increase yield in different waysdepending on the hybrid by pollen donor combination (Tables 3a and 3b).The first cross. H1 pollinated with P1, resulted in an 8.7% heavierkernel due to cross pollination, and 1.9% higher test weight. The secondcross, H2 pollinated with P2, resulted in kernels that had 2.8% highertest weight but were not heavier per kernel. The third cross, H2pollinated by P3, resulted in a 5.1% heavier kernel due to crosspollination, but test weight was not affected.

Tables 3a and 3b. The effect of self-pollination vs cross-pollinationvia practicing the invention on grain yield characteristics:

grain weight # of ears increase segregating yellow purple due Pollen foryellow kernels kernels to cross Hybrid donor and purple g/kernelpollination P Value H1 P1 32 0.331 0.360 8.7% <0.0001 H2 P2 11 0.3090.313 1.3% (NS) 0.2994 H2 P3 6 0.353 0.371 5.1% 0.0002

Test weight yellow purple increase kernels kernels due to cross Hybridg/volume pollination P Value H1 25.55 26.06 1.9% <0.0001 H2 26.52 27.272.8% 0.0004 H2 26.68 26.93 0.9% (NS) 0.3574

Example 3

An example of the invention described herein is the common situation incommercial corn grain production where there are large fields (20.2-40.5hectares (50-100 acres) per field or more) of different and unrelatedhybrids, in many case from different commercial vendors. These fieldswill largely self-pollinate and therefore will incur lower yield than ifthey were cross pollinated with an unrelated pollen source. On average,a pure stand of corn will contain greater than 95% grain at harvest thatis the result of self-pollination. Goggi et al. ((2006) Field Crops Res.99: 147-157) showed outcrossing between adjacent grain fields was farless than this: 0.4% at 35 m, less than 0.05% at 100 m.). Many of theselarge fields of corn will be of different maturities and thereforeflowering and shedding pollen at different times. In this example, thepollen from the earliest shedding field is collected andpreserved/stored until the second unrelated hybrid field begins toflower, and then pollen from the first hybrid field is applied at anearly morning hour (e.g. 5 am) to enhance cross pollination and minimizeself-pollination in the second hybrid field. The application isconducted for two to three days in a row during the heaviest floweringdays. The process is repeated by collecting, preserving, and storing thepollen from the second field and applying it to a third unrelated hybridfield when it begins to flower, and so on to a large number of fieldsuntil the flowering season ends. In this way, a significant portion ofthe corn grain in a region is the result of cross pollination ratherthan self-pollination, resulting in a yield increase in these fields of5-10%. Further, this process does not have to be exactly sequential asdescribed above. There would be adequate pollen from the first hybridfield (earliest to flower) to pollinate many fields (5 or more). In thisway, the cross-pollination that occurs as a result of this inventioncould spread very quickly from one field to 5 in the second set offields, to 25 in the third set, and so on. The result would be thatcross-pollination is enabled in a large majority of corn fields in anygiven area.

Example 4

Another example of the invention described herein is similar to example2 above in which pollen from unrelated hybrid fields that flower atapproximately the same time is harvested as soon as the first tasselsbegin to shed in each field. A portion of the pollen from each field isimmediately applied in a directed fashion to exposed silks in the otherfield, while the rest of the pollen is preserved/stored. The hybridfields will flower for approximately 3-4 days, and the stored pollen isused in the following 2-3 successive days to pollinate in the earlymorning prior to natural pollen shed to enhance cross pollination. Inthis way cross-pollination is increased and therefore yields in bothfields will be increased by 5-10%.

Example 5

In the specialty grain markets, it is very important that a field isallowed to entirely self-pollinate with very little cross pollinationfrom other nearby corn. One example is “food grade” corn, in which theremust be 99% selfing or more to meet the specifications (Strayer, D.(2002) Preserved Systems: A reference handbook. CRC Press.). In thisexample, the invention is utilized to decrease the amount ofcross-pollination to enable a pure harvest of the target specialty grainthat maintains the premium price with the end-user. In this case, pollenis harvested as soon as the first tassels begin to shed in the specialtygrain field. A portion of the pollen is immediately intentionallyapplied in a directed fashion to exposed silks in the field. There isonly a small percentage of silks that are exposed at this first stage offlowering. The rest of the harvested pollen is preserved/stored and isused in the following 2-3 successive days to pollinate in the earlymorning prior to natural pollen shed to enhance self-pollination andminimize cross-pollination. In this way, a specialty grain producerensures a successful harvest and delivery of specialty grain that meetsthe end user specifications. This example could also be practiced in aless intense fashion by only applying the pollen to the outer edge,approximately 12.2 m (40 feet), of the field where most of the crosspollination is most likely to occur, and allowing the inner part of thefield to pollinate naturally.

Example 6

This invention is applied to the corn grain industry in a way thatallows for producer decisions to be made based on markets right up tothe time of pollination (early to mid July) rather than makingproduction decisions prior to planting, as is currently the case.Through the collection and preservation/storage of many different pollensources, which are abundantly available in many different productionsituations globally, many different grain characteristics can bedelivered to a hybrid field via the pollen selected to pollinate. Aproducer could watch the markets and decide among a specialty grain,such as high oil, or no 2 yellow dent commodity grain, and could placean order for the pollen that will deliver the grain desired. In thisexample, the hybrid that is planted is simply a female receiver for thepollen, and has generally desirable characteristics. The pollen containsthe genes for the desired market and in this way a grain crop can bemodified up until the time of pollination to match a market need. Thereare many other similar traits that apply in this example. For instance,if a drought is being encountered, a pollen source could be used thatminimizes kernel abortion during grain fill. Alternatively, if theseason has not been water limiting, a pollen source could be used thatmaximizes sink strength and grain yield.

For the examples given above (high oil vs no 2 yellow dent commoditygrain) and many others, the characteristics could be achieved byutilizing naturally occurring, non-GMO pollen sources. For other traits,such as insect resistance in the grain, the desired characteristic couldbe achieved via a GMO source of pollen. Further, the current inventionenables the delivery of GMO grain characteristics without the commonconcern of negative effects found in conventional GMO plants. In thisway, this invention circumvents the massive hurdle that many beneficialGMOs have to get over to make it to market.

Example 7

A further example of the invention described herein is recovery of lostseed set under drought conditions that occur prior to or during thecritical flowering period. Drought stress during this time is by far thegreatest cause of lost grain production on an annual basis in the US.Under favorable conditions of adequate moisture and normal temperatures,there is close temporal synchrony between pollen shed from the maleflowers in the tassels and emergence of the pollen receptive tissues ofthe female flowers (silks). Under drought conditions, however, threeproblems develop that disrupt the success of pollination. First, droughtdelays silk emergence relative to pollen shed. (Basset, P. & M. Westgate(1993) Crop Sci. 33: 279-282) That is, the Anthesis-Silking Interval(ASI) increases, which can completely prevent pollination of lateemerging silks. Second, drought shortens the time emergent silks remainreceptive to pollen. (Araus, J. L. et al. (2012) Front. Physiol. 3: 305)That is, their capacity to support pollen germination and pollen tubegrowth necessary or fertilization is diminished by several days. Third,high temperatures and low humidities that usually accompany a droughtdecrease pollen viability and longevity. (Schoper. J. B. et al. (1987)Plant Physiol. 83(1): 121-125). To apply the invention to this case,fresh pollen is harvested within the field, or a nearby field of anearlier maturing corn hybrid. The pollen is optionally stored for aperiod of time, and then applied directly to the female flowers as theirsilks emerge. This application overcomes all three of the referenceddrought-based limitations on pollination and kernel set. The storedpollen can be applied on several successive days to enhance naturalself/sib pollination. In situations where drought causes the ASI toincrease to 4 days or more, application of the invention can increaseyields by 25% or more.

Although various representative embodiments of this invention have beendescribed above with a certain degree of particularity, those skilled inthe art could make numerous alterations to the disclosed embodimentswithout departing from the spirit or scope of the inventive subjectmatter set forth in the specification and claims. In some instances, inmethodologies directly or indirectly set forth herein, various steps andoperations are described in one possible order of operation, but thoseskilled in the art will recognize that steps and operations may berearranged, replaced, or eliminated without necessarily departing fromthe spirit and scope of the present invention. It is intended that allmatter contained in the above description or shown in the accompanyingdrawings shall be interpreted as illustrative only and not limiting.Changes in detail or structure may be made without departing from thespirit of the invention as defined in the appended claims.

Although the present invention has been described with reference to theembodiments outlined above, various alternatives, modifications,variations, improvements and/or substantial equivalents, whether knownor that are or may be presently foreseen, may become apparent to thosehaving at least ordinary skill in the art. Listing the steps of a methodin a certain order does not constitute any limitation on the order ofthe steps of the method. Accordingly, the embodiments of the inventionset forth above are intended to be illustrative, not limiting. Personsskilled in the art will recognize that changes may be made in form anddetail without departing from the spirit and scope of the invention.Therefore, the invention is intended to embrace all known or earlierdeveloped alternatives, modifications, variations, improvements, and/orsubstantial equivalents.

The invention claimed is:
 1. A method of grain production, comprising:a. growing a designated female plant that includes both female and malecomponents; b. intentionally cross-pollinating, on one or more days,said designated female plant with designated male plant pollen from adifferent genetic background when said male components are not sheddingpollen on said one or more days; c. growing the designated female plantto maturity; and d. harvesting the grain produced by said designatedfemale plant; wherein said male components are not male sterile at thetime of cross-pollination and said female components are not covered toprevent undesirable pollination.
 2. The method of claim 1 wherein saidmethod results in one or more of the following: a. Increased grainyield, and b. Modified grain characteristics.
 3. The method of claim 1wherein the designated male plant pollen is comprised of one or more ofthe following: a. Fresh pollen, and b. Preserved pollen.
 4. The methodof claim 1 wherein said designated female plant is any one or more of: acorn plant, a soybean plant, a wheat plant, a rice plant, a sunflowerplant, a canola plant, a sorghum plant, a barley plant, or a pearlmillet plant.
 5. The method of claim 1 wherein the intentionalapplication of designated male plant pollen is conducted by any one ormore of: mechanical means, pneumatic means, positive pressure means,negative pressure means, manual means, or combinations thereof.
 6. Themethod of claim 1 wherein said designated male plant pollen is freshpollen which has been harvested from one or more of a field, a growthchamber, a greenhouse, a glasshouse, a shade house, a hoop house, avertical farming facility or a hydroponic facility.
 7. The method ofclaim 1 wherein said designated male plant pollen is preserved pollenwhich has been previously collected and has been preserved by chilling,cooling, cryopreservation, freezing, freeze drying, or storage in liquidnitrogen.
 8. The method of claim 1 wherein said designated male plantpollen is applied on more than one occasion to the same designatedfemale plant.
 9. The method of claim 8 wherein said designated maleplant pollen is applied at the time which the female plant first becomesreceptive to said pollen.
 10. The method of claim 2 wherein the modifiedgrain characteristics include one or more of grain size, grain content,or grain composition.
 11. The method of claim 1 wherein said designatedmale plant pollen is obtained from sources optimal for application basedon environmental conditions.
 12. The method of claim 11 wherein saidenvironmental conditions are abiotic conditions.
 13. The method of claim12 wherein said abiotic conditions include at least one of drought,humidity, temperature, nitrogen availability or nutrient availability.14. The method of claim 11 wherein said environmental conditions arebiotic conditions.
 15. The method of claim 14 wherein said bioticconditions include at least one of insect pest pressure or diseasepressure.
 16. The method of claim 1 wherein said designated male plantpollen is obtained from sources optimal for application based on plantperformance data.
 17. The method of claim 1 wherein the designated maleplant pollen influences the characteristics of the grain harvested fromthe female plant, wherein said influence impacts one or more of: i.grain oil content; ii. grain starch content; iii. grain protein content;iv. grain oil composition; v. grain starch composition; and vi. grainprotein composition.
 18. The method of claim 1 in which said designatedmale plant pollen is obtained from a single genetic source.
 19. Themethod of claim 1 in which said designated male plant pollen is obtainedfrom multiple genetic sources and is combined prior to application. 20.A method of preventing undesirable pollination in grain production,comprising: a. growing a designated female plant that includes femalecomponents that receive designated male plant pollen; and b.intentionally pollinating said designated female plant on one or moredays with designated male plant pollen prior to said female plant beingsubjected to undesirable pollen on said one or more days; wherein saidfemale plant is not male sterile at the time of cross-pollination andsaid female components are not isolated to prevent undesirablepollination; and wherein said undesirable pollen has the same geneticbackground as said designated female plant.
 21. The method of claim 20wherein the step of intentionally pollinating said designated femaleplant occurs prior to said designated female plant pollen becomingviable.
 22. The method of claim 20 wherein the step of intentionallypollinating said designated female plant occurs at a time when saiddesignated female parent plant pollen is not shedding.
 23. The method ofclaim 20 wherein the step of intentionally pollinating said designatedfemale plant occurs at a time when said designated female plant pollenis not shedding or viable.
 24. The method of claim 20 wherein the stepof intentionally pollinating said designated female parent plant occursduring the morning.
 25. The method of claim 20 wherein the level ofcross-pollination is modified compared to natural pollination.